DE102014107342B4 - Device and method for detecting cancerous tumors and other tissue changes - Google Patents

Abstract

Method for spectrally analyzing a sample (100), comprising the steps of: attaching a cannula (20) to the sample (100), the cannula comprising at least a first optical fiber (34) and a second optical fiber (36), while the cannula (20 ) is attached to the sample (100): generating a first light pulse and directing the first light pulse through the first optical fiber, irradiating the sample (100) with the first light pulse through the cannula (20), reading out the spectral response of the first light pulse, generating a second a light pulse different from the first light pulse and guiding the second light pulse through the second optical waveguide, the second light pulse being a light pulse exciting a Raman light pulse of the sample,irradiating the sample with the second light pulse through the cannula (20),reading out the spectral response of the second light pulse,depositing the cannula (20) from the sample.

Description

field of invention

The invention relates to a method for spectrally analyzing a sample.

Background of the Invention

The healthcare market is one of the most important and profitable markets in the world today. The development is increasingly focused on the expansion of new diagnostic and treatment methods. A particular focus is placed on the development of cancer drugs.

Developing marketable products from innovations is one of the central tasks of various research groups in pharmaceutical and medical technology. In addition to the social and humanitarian aspects, efficiency and cost considerations are therefore playing an increasingly important role in health policy.

The classic methods of cancer diagnostics range from imaging methods such as scintigraphy and computer tomography to microscopic and immunological methods. To this day, a biopsy and subsequent diagnosis, based on microscopic images, by a pathologist is the gold standard in the diagnosis of tissue changes. Since the 1990s, research groups have published an increasing number of publications on cancer diagnosis using spectroscopy. The majority deal with Raman or fluorescence spectroscopy. The measurements were almost exclusively carried out ex vivo after special sample preparation.

Spectroscopy is commonly understood to mean analytical techniques that study the interaction of electromagnetic radiation with matter. It enables atoms, ions and molecules to be characterized by their typical wavelengths, which can be measured using emission, absorption, scattering, etc. Electromagnetic waves such as light, gamma rays, radio waves, microwaves, etc. can interact with matter by being absorbed, reflected or scattered. Today, spectroscopy is one of the most important instrumental analysis methods in physics, chemistry and astronomy.

Optical spectroscopy is usually understood to mean the use of electromagnetic radiation in the wavelength range from approx. 200nm to approx. 100µm for the qualitative and quantitative analysis of samples. Depending on the wavelength, different spectroscopy techniques are distinguished methodologically. First, ultraviolet and visible spectroscopy (UV/VIS) in the frequency range from 200 nm to 780 nm with the excitation of valence electrons. The ultraviolet spectral range (UV) extends from 200 nm to 370 nm and the visible spectral range (VIS) from 370 nm to 780 nm - and harmonics in molecules.

Finally, although not conclusively, mid-infrared spectroscopy (MIR) in the frequency range of typically 2.5 µm to 25 µm with the excitation of fundamental vibrations in molecules.

Photoluminescence is generally understood to mean the time-delayed emission of a light quantum after previous light absorption. Depending on the electronic transition or time delay, a distinction is made between fluorescence and phosphorescence. In fluorescence, the time difference between absorption and emission of a photon is typically on the order of 10 ^-9 to 10 ^-6 s, while in slowly decaying phosphorescence it is 10 ^-3 to 10 ^-5 s. The fluorescence is mostly stimulated by UV/VIS light.

One focus of Raman spectroscopy has so far been in the diagnosis of breast cancer. Specimen preparation ranges from snap-frozen, formalin-fixed, paraffin-embedded sections to unfixed frozen tissue.

The distinction between normal tissue, benign (non-cancerous) and malignant (cancerous) tumors in the intestine has also previously been examined ex vivo. For this purpose, the samples can be examined ex vivo after the biopsy, for example. The differentiation of lung carcinomas and normal tissue, the detection of squamous cell carcinomas in the neck and head area (hemotoxylin and eosin staining) and esophageal cancer are also already possible ex vivo. Another area of application is in the detection of precancerous lesions in dysplasia of the cervix, as a replacement for the so-called PAP test (Papanicolaou smear), i.e. the vaginal cytological smear (staining method of cells and assessment of their malignancy).

In ongoing research work, special attention is already being paid to near-infrared radiation (NIR) for optical examination, which is particularly suitable for examining tissue due to the high penetration depth (higher energy of this light, low absorption coefficient). Water, hemoglobin, lipids, proteins and DNA could be recognized here. However, no special marker substances have been identified so far, only wavelength ranges. So far, only qualitative statements have been possible for most substances.

The detection of pancreatic cancer and colorectal cancer has also been studied ex vivo. The analysis was always carried out on fresh biopsies without post-treatment.

Also with regard to the frequency ranges to be used, which require different measurement and evaluation methods, there are only a few concrete results so far. Due to the high material costs, the mid-infrared spectral range (MIR) has so far led a shadowy existence in experimental setups for in-vivo diagnosis.

The international patent application WO 01/97902 A2 describes a system with which images can be obtained in vivo in an endoscopic procedure. Identical light pulses are always sent into the area to be examined and different spectral components are evaluated. However, this application does not describe the use of specific different light pulses, and in particular also no investigation of a Raman shift.

Current diagnostic systems are also expensive and not very user-friendly.

General Description of the Invention

Marker substances are usually used to enhance the fluorescence response. However, it also seems possible to use endogenous “marker substances”. For example, hormones, enzymes, proteins and especially their increased occurrence in the blood or tissue can indicate a disease state. In contrast to conventional, injectable marker substances, the body's own "marker substances" do not have to be approved like drugs. The costs incurred here as well as the necessary animal experiments and, if necessary, experiments on human subjects can be omitted. The use of endogenous marker substances may make it possible to dispense with the use of complex and cost-intensive marker substances, and conclusions about various tissue states can be obtained quickly and inexpensively based on the concentration of the endogenous substances.

For example, nicotinamide adenine dinucleotide (abbreviated to NAD/NADH or coenzyme 1) is a hydride ion (two-electron/one-proton) donating coenzyme involved in numerous redox reactions of cell metabolism. It serves as an oxidizing agent and plays a central role in the breakdown of carbohydrates, for example. NADH is sometimes referred to as the "carrier of life energy". How essential NADH is to all life is shown by the fact that it is present in practically every human, animal or plant cell, so that the more active a cell is, the more NADH it uses. NADH is therefore also an interesting candidate with regard to photometry, since, in addition to its universal occurrence, it also shows distinguishable behavior with the tools of fluorescence. NADH shows an absorption maximum at a wavelength of 340 nm, in contrast to NAD. From this, conclusions can be drawn about the metabolic activity.

In addition to NADH, collagen crystallizes out as a marker substance. When evaluated correctly and intelligently, this fluorescence can be used as an important indicator of cell metabolism, the redox state and oxygen deficiency in the respiratory chain. An increase in the NADH concentration and thus its fluorescence indicates, for example, reduced tissue activity and the associated low NADH consumption. Conversely, a reduced NADH concentration in the tissue indicates an increase in metabolic cell activity. This fact can be used in the localization of tumors.

Hemoglobin is an iron-containing, oxygen-carrying protein found in the erythrocytes (red blood cells) of vertebrates. Oxygen transport occurs via binding to an iron complex of heme (protopophyrin °IX). The characteristic spectrum of oxygen-loaded (oxyhemoglobin) versus oxygen-free (deoxyhemoglobin) hemoglobin shows that the spectra largely overlap and conclusions about blood flow and oxygen saturation can be drawn directly from the course of the spectra.

The NADH/NADPH fluorescence is an important indicator of cell metabolism, the redox state, as well as oxygen deficiency and disturbances in the respiratory chain. An increase in the NADH concentration and thus its fluorescence indicates reduced tissue activity and the associated low NADH consumption. Conversely, a reduced NADH concentration in the tissue indicates an increase in metabolic cell activity. It is precisely this fact that is used in tumor localization; tumorous tissue exhibits reduced NADH fluorescence compared to normal tissue.

However, a measurement of the NADH fluorescence for a possible in vivo application in the Tumor localization in internal organs has so far not been possible due to the size of the measuring devices to be used.

Based on a platform technology (signal acquisition and communication), a flexible and miniaturized system is now to be presented, which is very robust against external influences.

It is therefore an object of the invention to be able to distinguish between tumorous and healthy tissue by means of a system. Tumorous tissue can be understood to mean malignant and benign tumors in the broadest medical sense.

It is also an object of the invention to be able to carry out different measurements at one point of the tissue in such a way that the same tissue is also covered by the different measurements.

Other objects will emerge from the following description or the particular advantages that are achieved with certain embodiments.

The object is solved by the subject matter of the independent claims. Advantageous embodiments are the subject matter of the dependent claims.

The method of spectrally analyzing a sample includes the following steps. First, a cannula is attached to the sample. For example, the cannula can be held by a displaceable analysis probe holder and attached to the sample by an electric motor. However, it is also possible for the cannula to be placed against the sample by hand.

The application to the sample can either be applied to the sample, with the cannula touching the sample over a large area with a measuring tip, or insertion into the sample, with the cannula penetrating, in particular piercing, at least with the measuring tip into the sample.

If the cannula is attached to the sample, a first light pulse is generated and the first light pulse is guided into the cannula. For example, the first light pulse can be guided via a first optical waveguide, which in particular is guided directly into the cannula. The first light pulse is advantageously generated while the cannula is attached to the sample and the cannula is not being moved. In other words, it is advantageous if the cannula, as far as possible, does not move relative to the sample.

The sample is then irradiated or illuminated through the cannula with the first light pulse. The first light pulse can be a coherent light bundle or an incoherent light bundle; Both types have advantages, which is why it is particularly preferred to use both types at the same time.

A second light pulse, which is different from the first light pulse, is also generated and guided into the cannula. The sample is then irradiated with the second light pulse through the cannula while the cannula is still attached to the sample.

The spectral response of the second light pulse is also read out, only then is the cannula removed from the sample.

In a preferred embodiment, while the cannula is attached to the sample, a third light pulse is also generated, which is different from the first and the second light pulse. The third light pulse is also guided into the cannula.

While the cannula is still attached to the sample, the sample is irradiated or illuminated through the cannula with the third light pulse. The spectral response of the third light pulse is also read out, after which the cannula is removed from the sample.

A multispectral analysis probe for analyzing different spectral ranges of a sample includes a cannula on a sample side of the multispectral analysis probe. The sample side is the side facing the sample.

Optical conductors can be encapsulated with the cannula. In a simple embodiment, the cannula corresponds to a sleeve or a cylindrical tube piece with an opening on the side facing away from the sample or with openings on both sides. On the side facing away from the sample, for example, optical conductors can be inserted through the opening in the cannula.

A measuring tip is located on the sample side of the multispectral analysis probe. In other words, the sample side of the multispectral analysis probe can be formed by a measuring tip. The measuring tip can either be a stopper or an attachment that is designed to allow light to pass through, so that a spectral observation of the sample through the measuring tip is made possible.

Light optics can also be used in the measuring tip. For example, the measuring tip be designed in such a way that it focuses the radiation that passes through it.

The measuring tip can also have a differently designed surface. The measuring tip can be flat. In particular, a surface of the sample can be scanned with a flat measuring tip. In other words, the flat measuring tip can be placed against a sample, ie in particular pressed, with the flat measuring tip not penetrating into the sample since it has a flat surface. Such a shape of the measuring tip is advantageous if, for example, the surface of the sample is to be analyzed, as is carried out in particular in the case of biopsies.
On the other hand, the measuring tip can be formed with a pointed tip, so that the measuring tip and is suitable for penetrating into the surface of the sample. In other words, in this case the measuring tip is ground, beveled or quite generally pointed or sharpened, so that a structure which tapers towards the tip is formed and which can penetrate the sample particularly easily. In particular, the measuring tip can have the shape of a needle tip and can be pierced into tissue in a manner similar to, for example, a needle for taking blood.

The multispectral analysis probe preferably includes a first optical waveguide for transporting a first light pulse. The optical waveguide is thus suitable for transporting a photonic pulse of a specific frequency range or a specific frequency from one location to another. In principle, plastic rods known to those skilled in the art are suitable for this purpose, into which the light waves can be coupled. Due to the better flexibility, however, flexible materials generally referred to as optical waveguides, in particular fiber-optic conductors, are preferred. In optical waveguides, light waves can couple in on a first side and couple out again on the second side, which is opposite the first. The most well-known representative of this type of fiber optic cable is a fiber optic cable. A quartz fiber line can also be advantageous depending on the frequency or spectrum used.

The multispectral analysis probe also includes a second optical waveguide for transporting a second light pulse, which is different from the first light pulse. The first and second light pulses are directed through the cannula to the probe tip to generate a spectral response on or in the sample. In other words, a light pulse is brought to the measuring tip and delivered to the sample in order to obtain a spectral response from the light pulse delivered to the sample.

To improve penetration into the surface of the sample, the measuring tip can be provided with a facet cut, a relief cut or a single cut. In other words, the measuring tip is ground so that the multispectral analysis probe can be inserted into the sample.

The outer diameter of the cannula is advantageously greater than or equal to 0.3 mm, preferably greater than or equal to 0.6 or 0.8 mm. The larger the outer diameter of the cannula, the easier it is to arrange optical waveguides in the cannula, or the more optical waveguides can be arranged in the cannula.

On the other hand, the outer diameter of the cannula is preferably less than or equal to 4.0 mm, more preferably less than or equal to 2.0 or even 1.5 mm. An upper limit for the outer diameter of the cannula is important, since on the one hand the X-rayed area of the sample can be limited and thus defined more narrowly. This is particularly advantageous when small tumors are to be observed, where healthy surrounding tissue would also be analyzed if the cannula was too large. The upper limit on the outer diameter of the cannula is also advantageous if the cannula is designed to penetrate the sample. For example, the cannula can penetrate tumorous tissue. Similar to an ordinary needle that is pierced into the skin, the cannula also has to displace the material of the sample, ie in particular the tissue, when it penetrates or pierces the sample. The displacement process creates a resistance to penetration, which is reduced with a smaller diameter of the cannula.

The wall thickness of the cannula must be large enough to withstand the load, especially the lateral "buckling load", when it is placed on the sample in order to protect the cannula or the multispectral analysis probe from breaking. On the other hand, a thin wall is advantageous for achieving a small outside diameter. The wall thickness of the cannula is preferably greater than or equal to 0.1 mm and less than or equal to 0.3 mm. The cannula can thus have a small outer diameter overall, but at the same time there is sufficient space inside the cannula for accommodating the optical waveguides.

The first light pulse and the second light pulse can be delivered to the sample via the cannula essentially simultaneously or in a short time interval one after the other. In other words, the first and the second light pulse are emitted to the sample with such a short time interval that the spectral response to the respective light pulse just barely corresponds to the associated light pulse can be assigned. In an advantageous embodiment, it is also possible to emit the first and second light pulses simultaneously or essentially simultaneously to the sample and possibly even to obtain only a common, superimposed spectral response. The aim is that the light pulses are essentially emitted onto the same sample material. The closer the time interval between the first and the second light pulse, the lower the probability that the sample or the multispectral analysis probe will move in the meantime, ie in particular that there will be a relative movement between the sample and the probe. On the other hand, depending on the design of the evaluation electronics for evaluating the spectral responses to the light pulses, it can be advantageous for the light pulses to be recorded and analyzed as separate pulses. If necessary, the light pulses are evaluated by different evaluation electronics and in this case can be fed to the respective evaluation electronics via optical waveguides.

In a particular embodiment, the multispectral analysis probe can include a light mixer, with the first optical fiber coupling the first light pulse and the second optical fiber coupling the second light pulse into the light mixer, and the first and second light pulses being passed on via a common optical mixer conductor through the cannula for delivery to the sample become. In other words, the light pulses are coupled into the light mixer in front of the measuring tip, in particular in front of the cannula, and are transmitted simultaneously or one after the other in the common light mixer conductor. In a simple embodiment, the light mixer can be arranged in the vicinity of the sources. However, due to the damping behavior observed, it has proven to be advantageous to arrange the light mixer on or near the cannula, so that the light can bridge the shortest possible distance between the light mixer and the measuring tip (sample).

The light mixer conductor is preferably inserted into the cannula, it preferably ends in the cannula directly at the measuring tip or possibly forms part of the measuring tip.

A cannula end of the first optical waveguide can be inserted into the cannula and, for example, end directly at the measuring tip. The first optical waveguide can also form part of the measuring tip. In other words, in this embodiment, the cannula end of the optical fiber is directly adjacent to the measuring tip, which is made of optically conductive material, for example. In the further embodiment, the first optical waveguide itself is guided up to the edge of the sample side of the cannula, so that the cannula end of the first optical waveguide directly touches the sample when the cannula is attached to the sample. If the cannula end of the first optical waveguide touches the sample directly, the number of optical interfaces in the cannula can be further reduced, since the light in the first optical waveguide can propagate to the cannula end and exit directly into the sample without running through other material.

The second optical waveguide can also have a cannula end, which is inserted into the cannula and ends directly at the measuring tip or forms another part of the measuring tip.

The multispectral analysis probe can also have a third optical waveguide for transporting a third light pulse that is different from the first and second light pulses. The third light pulse is also guided through the cannula to the measuring tip in order to generate a spectral response on or in the sample. In other words, with a third light pulse, a further, different spectral response is obtained, in particular from exactly the same point on the sample, so that superimposing the analysis results of the spectral response of the third light pulse with the analysis results of the spectral responses of the first and second light pulses may provide new, additional information about provides the sample.

The third optical waveguide of the multispectral analysis probe can also couple the third light pulse into the light mixer. In this embodiment, the first, second and third light pulses are passed through the cannula via the common light mixer fiber for delivery to the sample.

A fourth optical waveguide is preferably inserted into the cannula in order to conduct the spectral response to the first light pulse from the cannula. In other words, in addition to the optical waveguide(s) for supplying an optical signal to the sample, an optical waveguide for removing the spectral response from the sample is also accommodated in the cannula. There is preferably also an outgoing optical waveguide for each incoming optical waveguide.

A fifth optical waveguide can thus be inserted into the cannula in order to guide the spectral response to the second light pulse out of the cannula. A sixth optical waveguide can also be inserted into the cannula in order to conduct the spectral response to the third light pulse from the cannula.

The optical waveguides can preferably be used side by side in the cannula. In other words, the optical fibers of on the side opposite to the sample are fed parallel to each other and tightly packed as a bundle of conductors into the cannula, so that there is a narrow, space-saving bundle and the outer diameter of the cannula can be further reduced.

In a particular embodiment, the first, second, third, fourth, fifth and sixth optical waveguide and the light mixer conductor are used side by side in the cannula, so that the individual delivery of the light pulses can be carried out on the sample as an alternative to delivery through the light mixer conductor, without the multispectral Remove the analysis probe from the sample.

The first, second, third, fourth, fifth and sixth optical waveguide as well as the light mixer conductor can together form the measuring tip of the multispectral analysis probe with the respective end of the cannula. In other words, the cannula ends of the optical waveguides lying next to one another on the sample side of the cannula come to rest in this embodiment in such a way that the optical waveguides come into direct contact with the sample when the multispectral analysis probe is placed on a sample.

In a preferred embodiment, the optical waveguides, i.e. in particular the first, second, third, fourth, fifth, sixth optical waveguide and/or the light mixer cable, comprise fiber-optic cables, i.e. flexible fiber optics, into which optical light can be coupled and propagated along the fiber-optic cable. This is to be understood in particular as glass fiber lines or quartz fiber lines.

The light waveguides, ie in particular the fiber optic guides, in particular the first, second, third, fourth, fifth, sixth light waveguide and/or the light mixer guide, preferably have a diameter greater than or equal to 0.05 mm, in particular greater than or equal to 0.1 mm. In fact, it has been found in experiments to explore the principles of the present invention that these diameters allow a higher yield of the spectral response.

The optical waveguides, i.e. in particular the fiber-optical conductors, in particular the first, second, third, fourth, fifth, sixth optical waveguide and/or the optical mixer conductor, on the other hand preferably have a diameter of less than or equal to 1.0 mm, in particular less than or equal to 0.4 mm. This was found as a compromise in order to allow a reasonable small size of the diameter of the fibers with the highest possible yield of the spectral response, so that the overall outer diameter of the cannula does not become too large and the cannula is thereby impaired in its handling.

The multispectral analysis probe may include a slidable analysis probe mount for attaching the cannula and for positioning the cannula on the sample. In other words, this can be a linear system, for example in the form of a motor-driven slide on a rail, which can be moved on the rail for adjustment purposes.

The first optical fiber may include a source end for connecting the first optical fiber to a first light source. In other words, the optical fiber is directly connected to the first light source via its source end. As a result, the number of optical components can be further minimized and thus the transmission, ie the permeability of the light signal transmission to the cannula, can be further improved.

The second optical waveguide can also comprise a source end for connecting the second optical waveguide to a second light source.

Moreover, the third optical waveguide can also include a source end for connecting the third optical waveguide to a third light source.

In a preferred embodiment, the fourth optical waveguide comprises an output end for connecting the fourth optical waveguide to a first evaluation device. Here, too, the light-optical principle applies to place as few optical components as possible in the beam path in order to keep the light yield as high as possible. A direct connection of the fourth optical waveguide to the first evaluation device is therefore advantageous.

The fifth optical fiber can also have an output end for connecting the fifth fiber-optic cable to the first or a second evaluation device. In other words, the fifth optical fiber can be connected to the same evaluation device as the fourth optical fiber. This can be the case if the first evaluation device is set up to provide a number of signal inputs, so that a number of spectral responses can be processed with one evaluation device.

The sixth optical fiber can also have an output end for connecting the sixth fiber-optic cable to the first or a third evaluation device.

An analysis system with a multispectral analysis probe for analyzing different spectral ranges of a sample includes a first optical one Source for generating a first light pulse. The first optical source is preferably spaced from the cannula.

The analysis system with a multispectral analysis probe for analyzing different spectral ranges of the sample also includes a second optical source for generating a second light pulse, which differs from the first light pulse.

The analysis system also includes a cannula on a sample side of the multispectral analysis probe for accommodating optical conductors and a measuring tip on the sample side of the multispectral analysis probe.

The first optical source and the second optical source are connected to the cannula in order to send the light pulses to the measuring tip.

The first light pulse from the first optical source and the second light pulse from the second optical source are preferably delivered to the sample essentially simultaneously or in a short time interval one after the other via the cannula, so that the light pulses are delivered essentially to the same sample material.

In one embodiment, a light mixer is included, wherein the first optical waveguide couples the first light pulse and the second optical waveguide couples the second light pulse into the light mixer, and the first and second light pulses are passed on via a common light mixer guide through the cannula for delivery to the sample.

The analysis system may further include a first optical fiber having a source end, the source end of the first optical fiber being connected to the first light source.

The analysis system may also include a second optical fiber having a source end, the source end of the second optical fiber being connected to the second light source.

The analysis system can now also further comprise a third optical source for generating a third light pulse, which is different from both the first light pulse and the second light pulse.
In this embodiment, a third optical fiber having a source end is preferably further included, the source end of the third optical fiber being connected to the third light source.

The first optical source can be a fluorescent light source, for example.

The second optical source can be, for example, a visual light source, in particular a halogen light source.

Finally, the third optical source may be a near-infrared light source.

In a specific embodiment, the three light sources in the above embodiment are used together so that a fluorescent light source, a visual light source, and a near-infrared light source are used simultaneously. All three different types of light pulses from the different types of light sources can preferably be radiated simultaneously into the multispectral analysis probe for transmission to the sample.

The first optical source preferably emits light in the wavelength range from 300 nm to 400 nm.

The second optical source preferably emits light in the wavelength range between 300 nm and 1200 nm, in particular between 700 and 1200 nm.

The third optical source preferably emits light in the wavelength range between 1200 nm and 2200 nm.

In other words, the first, second and third light sources emit light pulses of different types to one another, since each light source emits in a different frequency range. However, it is also interesting and therefore preferred that, for example, the first light source emits incoherent light, while the second light source emits coherent light and the light pulses are of different types against this background.

If necessary, these properties of the light sources can also be changed during the irradiation process of the sample. It is essential that the radiation from the first light source differs from the radiation from the second light source, so that when analyzing the spectral response from the sample, different results can be extracted from the spectral responses and the results can be linked to one another.

This linking of the different results for the same sample, but using different types of light pulses, enables new insights into the nature of the sample analyzed in each case.

The analysis system can be designed in such a way that the third optical waveguide also couples the third light pulse into the light mixer and the first, the second and the third light pulse via the common light mixer conductor through the cannula for delivery to the sample.

The analysis system can also include a fourth optical waveguide inserted into the cannula in order to conduct the spectral response to the first light pulse from the cannula, in particular to the first evaluation system.

A fifth optical waveguide can also be inserted into the cannula in order to guide the spectral response to the second light pulse out of the cannula. Finally, a sixth optical waveguide can also be inserted into the cannula in order to direct the spectral response to the third light pulse out of the cannula.

The analysis system with a multispectral analysis probe can also include a first evaluation system for spectral and/or photometric analysis of the spectral response to the first light pulse.

The first evaluation system can be set up to also carry out a spectral and/or photometric analysis of the spectral response to the second and/or third light pulse.

The optical waveguides of the analysis system can comprise glass fiber lines or quartz fiber lines.

The invention is explained in more detail below using exemplary embodiments and with reference to the figures, in which identical and similar elements are sometimes provided with the same reference symbols and the features of the various exemplary embodiments can be combined with one another.

character list

• 1 eine Seitenansicht einer multispektralen Analysensonde,
• 2 weitere Ausführungsform der multispektralen Analysensonde,
• 3 Schnitt der Kanüle der multispektralen Analysensonde,
• 4 Aufsicht der probenabgewandten Seite der Kanüle,
• 5 eine weitere Ausführungsform der Kanüle,
• 6 Aufsicht auf eine weitere Ausführungsform der Kanüle,
• 7 Beispiel für eine Belegung der Lichtwellenleiter,
• 8 weitere Belegungsmöglichkeit für die Lichtwellenleiter,
• 9 Aufbau eines Lichtmischers,
• 10 eine Ausführungsform des Lichtmischers,
• 11 Überblick über ein Analysesystem,
• 12 eine weitere Form der multispektralen Analysensonde,
• 13 Analysensondenhalterung,
• 14 Analysensondenhalterung aus einer anderen Blickrichtung,
• 15 Intensitätsverläufe für eingestrahltes Licht,
• 16 beispielhafte Spektralantwort.

Show it:

• 1 a side view of a multispectral analysis probe,
• 2 further embodiment of the multispectral analysis probe,
• 3 cutting the cannula of the multispectral analysis probe,
• 4 Supervision of the side of the cannula facing away from the sample,
• 5 another embodiment of the cannula,
• 6 Top view of another embodiment of the cannula,
• 7 Example of an assignment of the fiber optic cable,
• 8th additional assignment option for the optical fibers,
• 9 construction of a light mixer,
• 10 an embodiment of the light mixer,
• 11 overview of an analysis system,
• 12 another form of multispectral analysis probe,
• 13 analysis probe holder,
• 14 Analysis probe holder from a different perspective,
• 15 Intensity curves for incident light,
• 16 exemplary spectral response.

Detailed description of the invention

1 shows a first side view of a multispectral analysis probe 10. Optical waveguides 30 are guided to the cannula 20 in a protected and shielded manner by a breakage and bending protection device 32. A retaining sleeve 22 is arranged in the area of the transition between the external optical waveguide 30 and the cannula 20 . In the present case, the holding sleeve 22 can fulfill several functions at the same time. On the one hand, the cannula 20 can be fastened to the holding sleeve 22, for example glued in or screwed in or fixed in some other way. The holding sleeve 22 thus increases the stability of the multispectral analysis probe 10. Furthermore, the holding sleeve 22 can increase the grippability of the multispectral analysis probe 10, since a solid object can be held in the hand in a more relaxed and steady manner than the cannula 20 or the optical waveguide 30. The Holding sleeve 22 can therefore improve the analysis results when using the multispectral analysis probe 10 by hand, since the probe 10 can be held more still.

On the other hand, the retaining sleeve 22 can also be used directly to attach the multispectral analysis probe 10 to a holder 12 (cf. 13 ) be trained. In the simplest case, the probe 10 can be held in place by means of a clamp. If necessary, the retaining sleeve 22 includes fastening means, in particular hooks or latching lugs, for fastening to the holder.

In the embodiment of 1 the cannula 20 is several centimeters, for example 5 to 8 cm long. Such a cannula 20 is particularly suitable for piercing or penetrating a sample 100 (cf. 11 ) suitable. However, only a short cannula 20 with a length of e.g. 0.2 to 1 cm is also provided for the external scanning of the sample 100.

2 shows a further embodiment of the multispectral analysis probe 10. The individual light waveguides, first light waveguide 34, second light waveguide 36 and third light waveguide 38, are connected at their source end to a light source 80 (cf. 11 ) connected to guide the radiation from the light source 80. The individual optical fibers of the fourth 40, fifth 42 and sixth 44 optical fibers are equipped with an evaluation system 70, 72 (cf. 11 ) connected in order to direct the spectral response to the respective light pulse to the evaluation system 70, 72 for the purpose of analysis.

The individual light waveguides 34, 36, 38, 40, 42, 44 are combined in a light mixer 50 in this embodiment. In other words, the light mixer 50 includes optics that enable light waves from different conductors to be coupled into a single conductor. At the sample 100 (cf. 11 ) facing side of the light mixer 50 then exits a light mixer line 45, which exchanges the light bidirectionally with the cannula 20 and further with the sample 100. In this embodiment, the cannula 20 can be particularly slim or have a small diameter, since only the sample end of the light mixer line 45 is possibly accommodated in it.

3 shows a section of the cannula 20, a measuring tip 24 being shown at the sample-side end, which occupies the sample-side end of the cannula 20 and forwards the light of the optical waveguide. For example, the measuring tip 24 can be a glass stopper. In the form shown, the measuring tip 24 is flush with the optical waveguides 36, 44, 46, so that a particularly good transition of the light waves from the guide to the measuring tip 24 and vice versa is made possible. Possible beam paths 26 of the beam bundles leaving the individual optical waveguides are outlined in the measuring tip 24 .

The measuring tip 24 on the sample side of the multispectral analysis probe 10 is in 3 executed flat. In other words, the measuring tip 24 has a cylindrical blunt surface. The measuring tip 24 is therefore particularly suitable for positioning the multispectral analysis probe 10 on a sample 100 (cf. 11 ) suitable, i.e. in particular for pressing onto the sample 100. With the measuring tip 24 shown, analyzes of biopsies in particular can be carried out or on samples 100 in general (cf. 11 ) which are not pierced.

4 shows a top view of the side of the cannula facing away from the sample of the same embodiment as 3 . Thus, a top view of the optical waveguides 34, 36, 38, 40, 42, 44, 46 is shown, which in this embodiment are arranged parallel to one another. In this embodiment, not only source conductors 34, 36, 38 (first optical fiber 34, second optical fiber 36, third optical fiber 38) and analysis conductors 40, 42, 44 (fourth optical fiber 40, fifth optical fiber 42, sixth optical fiber 44) are provided, but in parallel the optical mixer conductor 46 is also guided in the optical waveguide package and in the breakage and bending protection 32 . In other words, the sample 100 (cf. 11 ) are irradiated both with individual light waveguides 34, 36, 38 and with the combined light mixer guide 46.

5 shows another embodiment of the cannula. On the sample 100 (cf. 11 ) facing away occur, for example, the in 4 optical waveguides 34, 36, 38, 40, 42, 44, 46 shown (the optical waveguides 36, 46, 44 are shown, the other four optical waveguides are hidden behind them) into the cannula 20. The optical waveguides are routed through the entire cannula 20 and end on the sample side. In this form, the measuring tip 24 is ground, ie provided with a slanting grind, in order to improve the piercing of the cannula into the sample 100 .

In the form shown, the ends of the optical waveguides form part of the measuring tip 24. In other words, the optical waveguides 34, 36, 38, 40, 42, 44, 46 on the sample side are ground together with the cannula 20 in such a way that they form the measuring tip 24 form together with the rim of the cannula 20 and have direct contact with the sample 100 when the multispectral analysis probe 10 is placed on the sample 100 or pierced the sample 100. This embodiment has the advantage that there are no other light-optical components between the optical waveguides and the sample 100 that impair the transmission, for example through reflection or refraction. In this embodiment, on the other hand, the grinding angle is important with regard to the incidence and emission angle and the coupling capability of the light-optical pulses.

6 12 shows yet another embodiment of the cannula 20 of the multispectral analysis probe in a plan view. The optical waveguides 34, 36, 38, 40, 42, 44 and 46 are routed to the sample side of the cannula 20, where they form the measuring tip 24 together with the edge of the measuring tip 24. The measuring tip is ground so that piercing the sample 100 is relieved.

7 shows another example for the assignment of the optical fibers. In this example, the first light pulses are sent to the sample 100 by means of the first optical waveguide 34 (cf. 11 ) with the first light pulses radiating in the ultraviolet (UV) frequency range. The second optical fiber 36 directs second pulses of light to the sample 100, the second pulses of light being laser pulses. The third optical waveguide 38 conducts third light pulses to the sample 100, the third light pulses originating from a broadband, ie in particular incoherent, halogen light source 80. The fourth 40, the fifth 42, the sixth 44 and the seventh optical waveguide 48 forwards the spectral response to the respective light pulse to one or more evaluation systems 70. For example, the fourth optical fiber 40 may pick up a near-infrared (NIR) spectral response from the sample 100 . Furthermore, for example, the fifth optical waveguide 42 can record a spectral response as a “Raman pulse” from the sample 100 (observation of the so-called “Raman shift”). Furthermore, for example, the sixth optical waveguide 44 can pick up a spectral response in the visual range (VIS) from the sample 100 and transmit it to the assigned evaluation system 70, 72 (cf. 11 ) forward onto. Finally, the seventh optical waveguide 48 can record a fluorescence spectral response from the sample 100, for example. The importance of the individual measurement methods and the frequency ranges of interest for them will be discussed below.

The special advantage of using 7 The arrangement shown is that four different spectral responses can be evaluated with such a compact device as the multispectral analysis probe presented here. Because of the better observed signal-to-noise ratio of the spectral responses, individual optical waveguides are preferred to the use of the light mixer 50 in this embodiment.

8th shows another way of assigning the optical fibers. The measuring tip 24, at which the optical waveguides end, is shown symbolically.

In this embodiment, the first optical waveguide 34 is designed as a transmission fiber 34 . In other words, the first optical waveguide 34 transports the light pulses to the sample 100. The light mixer 50, by means of which the radiation from different types of light sources 80 can be coupled into the transmission fiber 34, is not shown but is preferably used.

In the sample 100, the spectral response is distributed over the entire solid angle range. Only the fraction of the spectral response that is covered by the solid angle range of the optical waveguide measuring the spectral response can be used to analyze the sample 100 . Therefore, in the preferred embodiment, the 8th three fiber optic cables each provided for recording the spectral response. In other words, the second 36, the third 38 and the sixth optical waveguide 44 lead the spectral response to the first light pulse away from the sample 100 and to the evaluation system 70, 72. In this example it is light in the visual frequency range (VIS). The fourth 40, fifth 42 and seventh optical waveguide 48 carry the spectral response to the second light pulse away from the sample 100 to the evaluation system 70. This is light in the near infrared range (NIR).

9 shows an exemplary structure for a light mixer 50. In the example, a transmission fiber 34 and a receiver fiber 36 are coupled into the light mixer line 45. FIG. A dichroic mirror or beam splitter 52 is arranged in the light mixer 50 . The course of the rays is then initially from above out of the transmission fiber 34 into the light mixer 50. A converging lens 54 is inserted at the transition into the light mixer 50, which in combination with the further converging lenses 54 can improve the transmission of the light mixer 50. Following the arrows 56, the beam path leads to the beam splitter 52, which deflects the light pulse in the direction of the light mixer line 45, where the light pulse ends up in the sample 100 after passing through the light mixer line 45.

The spectral response couples from the sample 100 into the light mixer line 45 and is coupled into the light mixer 50 via the converging lens 54 following the arrows 58 . In the light mixer, the frequency-shifted spectral response passes through the beam splitter 52 and is coupled into the second optical waveguide 36, by means of which the spectral response is finally routed to the evaluation system 70, 72.

10 shows an embodiment of the light mixer 50. The light mixer 50 has cylindrical lens tubes 60, in which a as in 9 Converging lens 54 shown is arranged. The beam splitter 52 is arranged in the central part of the light mixer 50, which is shown square. Connection means 62, in particular screw means such as for example for SMA connectors, FC connectors or SAM connectors, are attached to the lens tubes 60 for connecting the optical waveguides 30. A movable lens tube 66 with a lens tube adjustment system 68 makes it possible to detect different types of frequency ranges and/or different types of spectral compositions with the light mixer 50 .

11 shows an overview of a simple embodiment of the analysis system 9 with a multispectral analysis probe 10. One or more light sources 80 or lasers 80 can be controlled by a control computer 72 to output light pulses. The light pulses of the light source(s) 80 are transmitted via optical waveguide 30, for example via the first optical waveguide 34 and/or the second and third optical waveguides 36, 38, to the multispectral analysis probe 10 and through the cannula 20 to the sample 100. The case shown is a tissue sample. In other words, the multispectral analysis probe 10 can be used to carry out non-therapeutic measurements on extracorporeal samples 100, for example biopsies. Synthetic or engineered samples 100 may also be used and analyzed for research or analysis of non-living materials.

In response to the light pulse introduced into the sample 100, a spectral response, possibly with a frequency shift, is produced, which again passes through the cannula 20 of the multispectral analysis probe 10 and via optical waveguide 30, for example the fourth optical waveguide 40 and/or the fifth, sixth and/or seventh optical waveguide 42, 44, 48 to the evaluation system 70, in this case a spectrometer 70.

The spectrometer 70 can be controlled by the control computer 72 if necessary. The analysis result obtained with the spectrometer 70 can preferably be evaluated further on the control computer 72 .

12 shows the multispectral analysis probe 10 in a form already built for test purposes. The cannula 20 is attached to the retaining sleeve 22 . The optical waveguides 30 are guided through the breakage and bending protection 32 , through the cannula 20 to the measuring tip 24 .

13 shows an embodiment of an analysis probe holder 12, in which the multispectral analysis probe 10 is used. The probe holder 12 has a bearing 14 for the analysis probe holder, in the embodiment a pin 14, by means of which the probe holder 12 can be rotatably mounted in a recess. This allows the multispectral analysis probe 10 to be used at different angles. The probe holder 12 also has a linear system 16 to which the analysis probe 10 is attached in a linearly displaceable manner. In other words, the linear system 16 can attach the analytical probe 10 to the sample 100 by adjusting the linear system, preferably automatically by means of the control computer 72 . More preferably, the linear system 16 enables several measurement points to be recorded at different depths in the sample 100 by the sample being pushed into the sample 100 step by step and light pulses being emitted to the sample 100 at time intervals, correlated to the advance of the linear system 16.

The retaining sleeve 22 is pushed into the probe receptacle 18 of the probe holder 12 and can be fixed there if necessary, for example by means of a catch or a screw clamp 17.

14 shows another view of the probe holder 12 with the multispectral analysis probe 10 inserted. The probe 10 is fixed in the probe receptacle 18 by means of the screw clamp 17 . The optical waveguides 30 are ready for connection to the respective source 80 or the evaluation system 70 .

15 FIG. 12 shows an example of intensity curves that are obtained when the sample 100 is excited at a specific frequency. For example, an excitation frequency of 360 nm (dashed line) has proven to be advantageous in the course of the invention. At this frequency, a satisfactory spectral response could be obtained for the particularly interesting NADH.

16 shows corresponding fluorescence emission spectra. It turns out that, for example, the spectral response for the evaluation of NADH is to be expected at 460 nm.

Due to the know-how available at the University Hospital Mannheim and the Institute for Process Measurement Technology and Innovative Energy Systems, a "low-cost sensor" could be developed. In the field of diagnostics, for example, in various studies by the two institutes on which the present invention is based, MIR spectroscopy has shown strengths in in-vivo cancer diagnostics, which are expected to also be used in the areas of arteriosclerosis, leukemia, the detection of brain tumors, arthritis, BSE and Diabetes can be used.

On the one hand, fluorescence measurements with an excitation light source of 365 nm were used. The expected intrinsic fluorescence is expected at 450nm, with which NADH in particular can be detected in the tissue. NADH is an oxidizing agent in the body that should be present in both healthy and tumorous tissue. Advantageously, the measurement data can be evaluated without further pretreatment or calculation steps, since the energy spectra generated can be used directly for the evaluation in the case of a fluorescence measurement.

In an animal model (using a mouse as an example), the wavelength of 800 nm (isosbestic point hemoglobin) could be used in a promising manner to detect tumors of 100 solid consistency. The isosbestic point describes a wavelength in a system in which a reaction takes place, but the light is declining sorption does not change. In this case, changing the ratio of deoxyhemoglobin and oxyhemoglobin. Since hemoglobin is a strong absorber in the near infrared (NIR), this is taken as a measure of blood flow. The higher the concentration, the higher the blood flow through the tissue. In the fluorescence technique, the parameters collagen and NAD(P)H emerged as markers.

The measurement system 9 presented, which is able to distinguish tumorous from healthy tissue 100 based on the spectra of the tissue samples 100, is constantly being further developed, so that a complex and usually expensive pathological diagnosis can possibly be dispensed with in the future. In the event of a possible later use in the healthcare market, this means an effective reduction in hospital costs and the associated therapy and laboratory costs. The knowledge gained with the measuring system should be able to be used to continuously develop, certify and also make marketable a high-quality cancer diagnostic tool for clinical use.

First, it should be briefly outlined how tumorous tissue can be detected, for example, by means of spectroscopic examinations. On the one hand, it was found that tumorous tissue 100 is characterized by a higher proliferation (cell division) and an increased metabolism. These tissue properties can be recorded by measurement. However, classic tumor markers, such as those used in blood, are useless for the localization of tumorous tissue (possibly with the exception of PSA). Overall, the aim of this joint project is to set up and further develop the multispectral, in particular fiber-optic, measuring system 10 which allows conclusions to be drawn about the tissue condition and thus localizes tumorous tissue 100 .

In one example, NIR (near infrared), VIS (visual), fluorescence and Raman spectroscopy techniques are combined in a fiber optic probe 10 or puncture sensor 10 . Meaningful wavelengths/wavelength ranges are taken from the information from the optical measurements and combined in an evaluation algorithm that is implemented in software with a user interface and is installed on a conventional PC 72 or tablet.

In addition to the biopsy needle 20, the prick sensor with optical waveguides 30 based on sapphire fibers and with directly attached electronics is used to achieve short fiber paths. A MIR-in-vivo-measurement is thus possible for the first time. The innovative sensor technology with the associated software enables a preliminary diagnosis in the body and/or a targeted biopsy. With this measuring equipment 9, a simultaneous in-vivo measurement of the tissue metabolism, its composition as well as the pH value, water content and O ₂ saturation can be detected for the first time and is open to evaluation. The number of biopsies at different locations could be reduced in the future and thus the costs on the diagnostic side could be significantly reduced. Depending on the performance of the system, the aim is to avoid a biopsy. The system will be able to recognize blood vessels and be available to the attending physician as an early warning system, which can prevent unforeseen bleeding.

As soon as the analysis system 9 has been further developed, in particular approved, with regard to the certification process to combine diagnosis and photodynamic therapy (PDT), biopsy and therapy are limited to just one intervention.

PDT is a treatment method for treating tumors and other tissue changes. Here, the light-activatable substance (photosensitizer) is stimulated by light of a specific wavelength. Toxic substances are formed that damage the tumor or tissue. The introduction of so-called seeds via the biopsy needle is also possible (therapy for prostate carcinoma). Seeds are radioactive metal pins a few millimeters in size. These are injected into the tumorous tissue and destroy the surrounding tissue. This inevitably leads to shorter operation times and shorter patient stays. This means an effective reduction in hospital costs and targeted therapy.

Since this innovative concept reacts highly sensitively to differentiate between fibrous (inflamed) tissue 100 and tissue with an increased proportion of fat, two additional fields of application are conceivable: diagnosis of fatty liver and recognition of the current liver status in the course of chronic hepatitis.

The presented multispectral analysis probe 10 is also of interest for basic research. Placed in the vicinity of tumorous tissue, conclusions can be drawn about the effectiveness or the influence of chemotherapy on the local metabolism. Direct influences of the drug on the cell metabolism during therapy become visible.

An important part of the measuring apparatus is the probe 10, for which a possible preferred material structure with the respective reference sources is given below. 7x1.40m glass fibers with a core diameter of 200 µm each, for example from Edmund Optics GmbH, can be used as optical waveguides. The fibers are guided, for example, through a standard cannula (1.1×50 mm Sterican 19G from B.Braun Melsungen AG) and end flush with the tip. In addition, connectors (multimode SMA905 connector for 200pm fibers) from Thorlabs GmbH are advantageously attached to all seven glass fibers. The fibers themselves are protected from the environment by seven protective sleeves (FT038-BK) from Thorlabs GmbH, since glass fibers are very sensitive to external forces.

Due to the lower refractive index of the inner fiber core compared to the cladding, total reflection occurs in the fiber and it is possible for waves or signals to be conducted through the fiber (general for optical waveguides).

The probe 10 is attached to the probe holder 12 in order to enable punctures at different angles. A round aluminum material 14 was cut to size and mounted as a bearing 14 for the probe holder in the center of the spindle attachment point. The central position is therefore chosen in order to be able to keep the leverage as small as possible. The round material can thus be easily attached to the laboratory stand with the laboratory clamp.

In order to be able to allow different puncture depths in the same puncture channel in the sample 100, one possibility is to couple the probe 10/cannula 20 to a linear system 16 in order then to be able to penetrate the tissue 100 step by step as precisely as possible. For example, in the case of the 13 a linear system from Spindler & Hoyer (now QOptics) to which the probe 10 is attached and adapted.

To attach the probe 10 to the spindle 16, an aluminum rail 18 is sawn to size, in which the probe 10 can be fixed in the middle with a lockable screw 17. The choice of aluminum is cost-effective and allows for easy processing and handling.

14 shows the structure of the measuring system 9 of the university. In this case, all the required measuring devices 70 are set up and connected to one another with the probe 10 via the optical waveguide 30 . In addition, the home-made probe is shown, which is adapted to the linear system 16 and attached to a laboratory stand so that it can then pierce the tissue sample 100 . The measured values are then converted into usable information in the measuring computer 70 .

The home-made probe 10, which in this embodiment comprises seven optical fibers 30, is inserted into the tissue, and it can first be determined which optical fiber (if the central transmission fiber is selected) supplies the best measured value. Since all results are directly related to each other, a good measurement signal can typically be achieved with each fiber.

Individual types of fabric may differ in their composition and optical properties. It was therefore important in the context of preliminary investigations to cover as broad a spectrum as possible and to define the measurement parameters.
Several tissue types (muscle, liver, fat, brain) are examined and their remission and extinction determined.
Different measuring points provide different deviations since the tissue 100 has a locally different tissue structure/composition.

Measurements are now initially made in the VIS range. For the purposes of adjustment and system diagnosis, the full range of wavelengths is used (as far as the system, consisting of silicon detectors (approx. 300-1100nm), allows) in order to cover the largest possible spectral range per measurement. This is followed by measurements in the NIR range. All measurements are performed with the same probe with the middle fiber as the transmit fiber. The assignment of the receiver/detector input to fibers 1-6 VIS-NIR is changed for adjustment and system diagnosis purposes (after a previous check, all fibers deliver similar measured values). In addition, with the VIS measurement, it must be noted that an opaque protective cover encloses the apparatus in order to keep out ambient light and thus exclude extraneous light as a fault. This is not the case with the NIR measurements, since the spectrum there lies beyond visible light, but the apparatus is also surrounded by a dark material.

Before starting the measurements on the tissue, a reference spectrum is first determined by measuring a white standard (e.g. available from HoffmanSphereOptics) at a distance of 4 mm from the cannula (reference). This distance was determined by various tests in such a way that the integration time and thus the measurement signal in the later tissue is sufficiently high. This is followed by the dark measurement by screwing a black cap or a metal cap (e.g. a brass cap nut) over the SMA input of the detector at the receiver input.

During the actual measurement process, the apparatus 9 is attached to a stand so that the linear system 16 can be used. Here, the various tissue samples 100 are now placed under the apparatus 9 in order to then be able to carefully pierce them with the measuring probe 10 . Each tissue 100 is now measured three times at different measuring points and then the mean value of these different punctures is calculated for further analysis. Now, based on the references (white standard), the remission and from it the extinction can be determined and displayed visually as diagrams. For example, it can be seen in this way whether the curves are largely superimposed or whether there are deviations that can be attributed in particular to a different composition of the tissue at the different puncture sites or measurement points.

In the fluorescence measurement, the tissue to be examined is irradiated or excited (NADH) with a wavelength of 365 nm, so that the tissue has an intrinsic fluorescence at 450 nm. This can be interpreted based on the course and the height of the peak. Since the measured samples come from the butcher and no longer show any metabolic activity, the deflections are very low, as expected. Metabolism products (particularly NADH) are largely degraded. However, it can be seen that liver appears to be the best absorber. The main absorbers in tissue are hemoglobin and water

Like the multispectral analysis probe 10, the UV light source 80 is a self-construction with a UV light-emitting diode with an emission frequency of 365 nm. The two spectrometers 70 are from Zeiss. On the one hand the Zeiss VIS/NIR spectrometer (MCS621/MCS611) which detects the NIR and VIS measurements and on the other hand a Zeiss CCD/UV-NIR spectrometer for fluorescence emission measurement. The suppression of extraneous light is realized with a simple cloth that envelops the tissue sample in order to keep out extraneous light sources that may interfere with the measurement.

For example, the following function parameters were used: VIS light pulse, duration 300 ms, accumulation 50; NIR light pulse, duration 200 ms, accumulation 50; Fluorescence time 6500 ms, accumulation 10.

A test animal, for example a mouse, can be used for test and experimental purposes for evaluating the analysis system 9 presented. B16 melanoma cells, which are known to spread and metastasize widely, can be injected subcutaneously, meaning the melanoma should be just under the skin. Malignant melanoma, a neoplasm (new tissue) from degenerated melanocytes (pigment cells of the skin), is one of the most malignant tumors of the skin and mucous membranes. Melanoma cells are therefore skin cancer cells that are placed directly under the skin's surface. Muscle tissue (hind leg) and subcutaneous tissue from the same test animal serve as reference tissue for our measurements.
The position of the subcutaneous tumor is first fixed in a stable lateral position on a base plate and punctured. Here, spectra are determined directly in the center of the tumor as well as 1 mm from the center. Subsequently, spectra of the reference tissues are recorded under the same measurement conditions. Muscle tissue on the hind leg and subcutaneous tissue were used for this purpose, for example.

Both forms of hemoglobin occur in the tissue to be examined. The spectra of oxy- and deoxyhemoglobin can overlap. For example, the following two conclusions can be drawn from the spectra of the various tissues: Firstly, how strongly the respective tissue is supplied with blood and secondly, how high the oxygen saturation of the respective tissue is.

A statement about the blood flow can easily be made from the peak height. In this case, muscle tissue is supplied with blood the most, and subcutaneous tissue the weakest, which can already be seen visually from the red coloring of the muscle tissue. In contrast, subcutaneous tissue is more translucent, jelly-like. Here, the tumorous tissue 100 assumes a middle position at both examined locations.

The local oxygen saturation of the tissue can be determined based on the properties of oxy- and deoxyhemoglobin in the peak at approx. 560 nm. Oxyhemoglobin has two maxima here, while deoxyhemoglobin has a maximum exactly between the two maxima of oxyhemoglobin. The peak shape allows qualitative statements to be made about the local oxygen saturation of the tissue. Oxygen saturation in tumorous tissue is significantly lower than in muscle and subcutaneous tissue.

By developing a mathematical algorithm, absolute values of saturation can be calculated from the spectra afterwards. The more pronounced the two maxima of the saddle are, the more oxygen-saturated the hemoglobin is. With high oxygen saturation in muscles or subcutaneous tissue, the peaks of oxy-hemoglobin are much more pronounced.

The near-infrared spectrum is dominated by the water bands at 960 nm second harmonic and 1450 nm first harmonic. The height of the peaks is a direct measure of the local water content of the tissue at the probe tip 24. In the tumorous tissue 100 the water content is a factor of two below that of the healthy tissue 100. The highest water content is in the muscular tissue, subcutaneous tissue is minimally below it .

In the case of fluorescence spectroscopy, the peaks after the actual excitation are of interest, since the metabolic activity (NADH, collagen) of the examined tissue can be recognized from them. These can be increased in tumorous tissue compared to healthy tissue. This suggests decreased tissue activity.

Depending on the performance of the system, the aim is to avoid a biopsy. The system is being further developed in such a way that it can recognize blood vessels and be available to the attending physician as an early warning system, which can prevent unforeseen bleeding. In addition, the doctor is visually informed about the current tissue composition and concentration. This information makes it possible to combine diagnostics and therapy in one operation. It is conceivable, for example, to first measure with the probe through a thicker cannula, and if diseased tissue is detected, to inject a so-called seed (radioactive metal rod, which kills the surrounding tissue) through the same cannula into the tissue, e.g. in the case of prostate cancer.

Since this innovative concept could also be used to differentiate between fibrous (inflamed) tissue, but also reacts sensitively to tissue with a high proportion of fat, two additional fields of application are conceivable for future projects: diagnosis of fatty liver and detection of the current liver status in the course of chronic hepatitis.

In addition, the sensor can also be placed in the vicinity of tumorous tissue, from which conclusions can be drawn about the effectiveness of chemotherapy on the local metabolism. Furthermore, direct influences of different medications on the cell metabolism can become visible during the therapy.

Optical information that can potentially be used for cancer detection via corresponding sensors 10 is distributed over a number of different types of spectral ranges. Cancer itself, i.e. cancer cells 100, cannot be directly distinguished spectrally from other cells. Combining different but narrowly defined spectral information, together with the knowledge of a medical professional, typical tissue changes associated with cancer 100 can be measured and inventively interpreted and detected. Surprisingly, it was found that the combination of certain properties measured is likely to indicate certain types of cancer.

For example, a fiber optic narrow probe 10 can penetrate tissue by puncturing. The basic design of the probe 10 will in most cases be that glass fibers are arranged inside a cannula 20, for example a stainless steel cannula 20. The design of the glass fibers 30 and the arrangement are subject to specific laws.

A regularity results from the application. The probe 10 to be built should be designed in such a way that it can easily be used to penetrate, preferably pierce, tissue. Various cut patterns are available, such as a bevel of less than 45°. Other cut patterns such as bevels in 2 directions are also an integral part of the invention. The cannula must accommodate the glass fibers 30. The cannula should therefore have an outside diameter of 0.3 to 4 millimeters, preferably 0.6 to 2 millimeters, more preferably 0.8 to 1.5 millimeters. The wall thickness of the cannula should be between 0.1 millimeters and 0.3 millimeters in order to create sufficient space inside the cannula 20 to accommodate the glass fibers 30. The laws on which the invention is based mean that coherent light sources 80 and non-coherent light sources 80, ie in particular different types of light sources 80, are both used. Coherent light sources 80 can be focused into very thin optical fibers 30 with a small cross-section or diameter. Incoherent light sources 80, on the other hand, provide light output at the probe tip that is proportional to the area of the optical fiber 30. Therefore, the glass fiber 30 should not be too small. However, because of the steric hindrance during the puncture and the flexibility of the overall system, the glass fiber 30 must not be too large either. A diameter of 50 micrometers seems to have emerged as the lower limit for the incoherent light sources. We see 1 millimeter as the upper limit; glass fibers 30 in the range from 100 μm to 400 μm are preferred. In practical tests, a current optimum was found with glass fibers with a diameter of 200 micrometers. The light of different wavelengths is at one end of the optical fiber 30 by the light source 80 - a laser 80, an LED 80, a broadband radiator 80 or other light source 80 - in the Optical fiber 30 is coupled in via optics, transported via the optical fiber 30 with the lowest possible attenuation, and is coupled out of the optical fiber 30 again on the probe side. Due to the slanted bevel, it must be taken into account that refraction and reflection can occur here. A special embodiment of the probe tip 24 is that the fibers 30 - for example glass or quartz fibers - come to lie next to each other and the direct optical path from the transmitter to the receiver is built in via layers lying in between.

The fact is that, in addition to the fibers that are suitable for sending light, some are also installed that are suitable for receiving light. Due to the lack of direct coupling, extremely weak signals can be measured. The developer of optical measurement technology is aware that any boundary layer on which light from the transmitter part of the device impinges, although this light is usually mainly transmitted, it is also reflected to a small extent or diffusely scattered. Arrangements that allow such scattering directly into the receiving fibers without product contact are subject to special care, as will be described later.

In another embodiment, there is a light mixer 50 at the probe tip 24 - for example a thicker piece of fiber with a diameter of one millimeter or another diameter, for example, into which the different light sources are coupled and emerge as mixed light at the probe tip 24.

On the reception side, the glass fibers 30 are supplied with light by the human or animal tissue 100 . The light from the tissue 100 is generated via scattering or re-emission at the radiated wavelength or an offset wavelength and reaches the receiving glass fiber 100. Concentrating optics 54 can be used here, but it can also be beneficial to the concentrating optics 54 entirely in other applications be waived. In one embodiment, the light then reaches the beveled glass fiber 30 , which comes to lie parallel to the transmission fiber 34 . The light with low attenuation is conducted via this glass fiber 30 to the detection and processed there.

For example, PIN diode circuits with known transimpedance amplifiers or other amplifiers can be used in the verification. Spectrometer systems consisting of CCD lines, PIN diodes or CCD surfaces can also be used, or other types of detectors such as multipliers, avalanche diodes or the like.

The simultaneous, parallel or sequential use of coherent and/or incoherent light and the integration of special knowledge about tissue compositions leads to new insights and new analysis methods using the presented multispectral analysis probe 10. It can be assumed, according to the experiments on which the invention is based, that The presence of cancer cells means that there is an altered metabolic system. This can lead to increased concentrations of certain metabolic intermediates such as NADH, protoporphyrin, FAD or other typical substances associated with metabolism. In a logical manner, one of the possible partial pieces of information within the scope of the invention is provided by specifically querying these metabolic intermediates. A laser 80 or a light-emitting diode 80 in the UVA range or in the short-wave blue range can be used for this partial task. A blue or green LED 80 or another light source 80 for this wavelength range could also be used for protoporphyrin.

Another connection found within the scope of the invention is the change in the local connective tissue density in the cancerous growth compared to the surrounding matter/material system tissue. This change changes the so-called elastic light scattering, i.e. the scattered light, which is scattered back again at the same wavelength. If you want to measure this part property, you move the measurement, for example, to a wavelength range in which few types of tissue show absorption. It proved advantageous to work in the long-wave visible range, such as red, or in the near-infrared range in the part of the spectrum that is not affected by water. This is the spectral range between 700 and 1,200 nanometers.

For technological reasons, it may appear advantageous to further restrict the range to the detection range of silicon, since silicon receivers are advantageously inexpensive and more sensitive to build than other near-infrared detectors. This range would then be limited to the long-wave end at 1100 nm.

However, it may also turn out that certain cross-sensitivities in the tissue make it necessary to switch to the 900 to 1,200 nanometer range, which means that indium gallium arsenide, for example, can be used as an electronic receiver. The elastic light scattering shows the density of the tissue for the part of tissue located directly in front of the probe.

Another aspect that surprisingly came to light in the course of the investigations is the change in the local water concentration in the tissue. The water concentration can be measured with the probe in a suitable manner if an incoherent radiator is used on the transmission side in the range of absorption of a water band in the near infrared, such as in the range of 1,200 nanometers or in the range of 1,400 nanometers or in the range of 1,900 Nanometer and on the detection side 1-3 wavelengths or more wavelengths are detected, which show an absorption of the water and generate a reference value with one of the other wavelengths. The reference value is typically taken at a wavelength at which the water does not absorb as much or not at all and gives a measure of the penetration depth of the light. The absorbed wavelength is related to this. In the simplest evaluation case, the quotient of these two wavelengths is evaluated, or the logarithm of the quotient, or another mathematical evaluation rule. It initially shows a still falsified water content. If it is recognized from parallel experiments on sampling and laboratory methods for cancer detection that the absolute water content is necessary, an absolute value can be calculated from the measured relative water content signal using a calibration function from elastic light scattering, penetration depth of the light cone and extinction.

Another important piece of information can be obtained from the local oxygen content in front of the probe. This can be measured by evaluating a spectrum or at least several wavelengths in the visible light range via the oxygen saturation of the blood. In principle, the oxygen saturation of the blood and its measurement are known. A correlation with other measurement ranges and a detection of cancer are not yet known. Similar to the near-infrared range, broadband light can be radiated in via a fiber 30 and detected via a parallel fiber 30 . In one embodiment, the detection of the near-infrared information and the detection of the oxygen measurements, which usually take place in the visible spectral range, can take place via the same fiber 30 . Either a combined spectrometer unit could be connected downstream on the detection side, or the various detectors 70 could also be operated via a beam splitter system. In the case of rapidly growing tumors, it is also known that the oxygen saturation is very low and the oxygen consumption is very high. Surprisingly, it was found that such a greatly increased metabolism takes place inside fast-growing tumors that it leads to anaerobic metabolism. This could be measured by measuring lactate, which could be detected using Raman spectroscopy and correspondingly exciting lasers.

For the particularly reliable detection of cancer, it was found that the evaluation of the Raman signal, for example to detect lactate concentrations that are different from zero, is of great importance. It is therefore advantageous to also radiate in a Raman light 80 to be excited via a laser. The Raman spectrum or at least several wavelengths from it can be integrated in the detection.

The visible measurement can also be similarly extended to the UV range depending on the cancer being examined.

The design of the probe can scan specific areas in the tissue 100 via a special design. If one chooses the embodiment of parallel fibers, the area directly in front of the probe, for example the first 50 microns, is not seen. Rather, the light is coupled into the tissue and the first overlap with the reception cone of the detection fiber is deep in the tissue, ie 50 micrometers away, for example. The optically skilled engineer can easily adjust the desired first overlap through the transmit cone and receive cone by the spacing of the transmit fiber and the receive fiber. The farther the transmitting fiber and receiving fiber are from each other, the lower the signal, but the deeper the scanning plane.

In the other direction, the extreme is that the transmission fiber and reception fiber have a 100% overlap, which can be implemented by beam splitting on the device side. With a beam splitter or a beam splitter cube, or with a similarly functioning device, the transmitter can be attached from one direction and the receiver offset by 90° or another angle. A part of the incident light is reflected, goes into the fiber, gets out of the probe at the tip of the probe and into the tissue. The light remitted at the same point or also fluorescent light or Raman light is coupled back into the same fiber, transported back into the beam splitter cube or another beam splitter and passes partly or completely through this into the detector system. In this embodiment, the overlap between transmitter and receiver is 100%, the end ring depth is virtually zero. Measurements are then taken directly at the interface between the fiber and the tissue. Depending on the application, one or the other embodiment can be cheaper. With split fibers there is less crosstalk from transmitter to receiver and end face contamination is not as critical as in the embodiment where transmitter and receiver have 100% geometric overlap.

The requirements for the verification page are specified by the respective application. As already explained, photometers and spectrometers can be used. With photometers, only individual wavelengths are detected, with spectrometers, many wavelengths are detected. On the other hand, the detection sensitivity of spectrometers is limited compared to photometers.

All information is preferably used at the same time for the evaluation. It has been shown that, for example, the measured Raman and fluorescence intensities depend on the intrinsic color of the tissue in the same condition of the tissue, i.e. cancer or not. Darker tissue absorbs the incoming radiation, so the signal is lower. It is therefore advantageous to also measure the inherent color of the tissue and to incorporate the corresponding evaluation into a correction.

All existing information on tissue changes can be included in the evaluation, for example via a correlation matrix that is to be developed for each type of cancer. So in particular Raman, fluorescence, remission spectra in the visible UV and near infrared. In individual cases, individual pieces of information may not be relevant. Then the designer of the probe can decide whether the receiving channels and transmitting channels should nevertheless be integrated into the device or left out for cost reasons. The alternative is simply not to consider the information in the evaluation.

It could be shown that this special combination of the wavelength ranges mentioned and a specific search for metabolic intermediates and local conditions such as water content, connective tissue density and metabolic observation and its level, detects the cancer directly from the nail tip with a very high probability. The evaluation can be completed so quickly that a quasi-continuous signal is generated along the stub path.

It is obvious to the person skilled in the art that the embodiments described above are to be understood as examples and that the invention is not limited to these, but can be varied in many ways without departing from the scope of protection of the claims. Furthermore, it is evident that the features, regardless of whether they are disclosed in the description, the claims, the figures or otherwise, also individually define essential components of the invention, even if they are described together with other features.

Reference List

9

analysis system

10

multispectral analysis probe

12

Analysis probe holder

14

Storage of the analysis probe holder

16

linear system

17

compression fitting

18

probe recording

20

cannula

22

retaining sleeve

24

measuring tip

26

beam path

30

optical fiber

32

Break and bending protection

34

first optical fiber

36

second optical fiber

38

third optical fiber

40

fourth optical fiber

42

fifth fiber optic cable

44

sixth optical fiber

45

common light mixer leader

46

light mixer ladder

48

seventh fiber optic cable

50

light mixer

52

beam splitter

54

lens, converging lens

56

Arrow

58

Arrow

60

lens tube

62

fasteners

66

moveable lens tube

68

Lens tube adjustment system

70

Evaluation system, spectrometer

72

Analysis and/or control computer

80

optical source

100

sample

Claims (26)

A method for spectrally analyzing a sample (100), comprising the steps of: Attaching a cannula (20) to the sample (100), the cannula comprising at least a first optical fiber (34) and a second optical fiber (36), while the cannula (20) is attached to the sample (100): Generating a first light pulse and directing the first light pulse through the first optical waveguide, Irradiating the sample (100) with the first light pulse through the cannula (20), reading out the spectral response of the first light pulse, Generating a second light pulse different from the first light pulse and directing the second light pulse through the second optical waveguide, wherein the second light pulse is a light pulse that excites a Raman light pulse of the sample, Irradiating the sample with the second light pulse through the cannula (20), reading out the spectral response of the second light pulse, Detaching the cannula (20) from the sample. Method according to the preceding claim, wherein the step of irradiating the sample (100) with the second light pulse through the cannula (20) is followed by the steps while the cannula (20) is attached to the sample (100): Generating a third light pulse different from the first and the second light pulse and guiding the third light pulse into the cannula, irradiating the sample (100) with the third light pulse through the cannula (20), reading out the spectral response of the third light pulse, Detaching the cannula (20) from the sample (100). Multispectral analysis probe (10) for analyzing different spectral ranges of a sample (100), comprising: a cannula (20) on a sample side of the multispectral analysis probe (10) for housing optical conductors (30, 34, 36, 38, 40, 42, 44, 45, 46, 48), the cannula further comprising: a measuring tip (24) on the sample side of the multispectral analysis probe (10), a first optical waveguide (34) for transporting a first light pulse to the sample, a second optical waveguide (36) for transporting a second light pulse, which is different from the first light pulse, to the sample, the second light pulse being a light pulse exciting a Raman light pulse of the sample, wherein the first and second light pulses are directed through the cannula (20) to the probe tip (24) to generate a spectral response on or in the sample (100). Multispectral analysis probe (10) according to the preceding claim, wherein the measuring tip (24) is flat and is suitable for scanning a surface of the sample (100), or the measuring tip (24) being pointed and suitable for penetrating the surface of the sample (100). Multispectral analysis probe (10) according to any one of the preceding claims, wherein the measuring tip (24) is pointed and provided with a facet cut, a relief cut or a single cut to improve penetration into the surface of the sample (100). Multispectral analysis probe (10) according to any one of the preceding claims, wherein the outer diameter of the cannula (20) is greater than or equal to 0.3 mm, in particular greater than or equal to 0.6 mm or greater than or equal to 0.8 mm, and/or wherein the outer diameter of the cannula (20) is less than or equal to 4.0 mm, preferably less than or equal to 2.0 mm or less than or equal to 1.5 mm, and/or the wall thickness of the cannula (20) being greater than or equal to 0.1 mm and less than or equal to 0.3 mm, so that the cannula (20) has a small outer diameter overall, but at the same time there is sufficient space inside the cannula (20) to accommodate the optical conductor (30, 34, 36, 38, 40, 42, 44, 45, 46, 48) remains. Multispectral analysis probe (10) according to any one of the preceding claims, wherein the first light pulse and the second light pulse are delivered to the sample (100) essentially simultaneously or in a short time interval one after the other via the cannula (20), so that the light pulses are delivered essentially to the same sample material (100). Multispectral analysis probe (10) according to one of claims 3 until 7 , wherein a cannula end of the first optical waveguide (34) is inserted into the cannula (20) and ends directly at the measuring tip (24) or forms part of the measuring tip (24) and/or wherein a cannula end of the second optical waveguide (36) extends into the Cannula (20) is inserted and ends directly at the measuring tip (24) or forms another part of the measuring tip (24). A multispectral analysis probe (10) according to any one of the preceding claims, further comprising: a third optical fiber (38) for transport a third light pulse different from the first and the second light pulse, the third light pulse being guided through the cannula (20) to the measuring tip (24) in order to generate a spectral response on or in the sample (100). Multispectral analysis probe (10) according to any one of the preceding claims, wherein a fourth optical waveguide (40) is inserted into the cannula (20) in order to conduct the spectral response to the first light pulse from the cannula (20), and/or wherein a fifth optical waveguide (42) is inserted into the cannula (20) in order to conduct the spectral response to the second light pulse from the cannula (20), and/or a sixth optical fiber (44) being inserted into the cannula for guiding the spectral response to the third light pulse from the cannula (20). Multispectral analysis probe (10) according to the preceding claim, wherein the first, second, third, fourth, fifth and sixth optical waveguide (30, 34, 36, 38, 40, 42, 44, 48) and an optical mixer guide (45, 46) are used side by side in the cannula (20), see above that the individual supply of the light pulses can be carried out on the sample as an alternative to the supply through the light mixer conductor (45) without removing the multispectral analysis probe (10) from the sample (100) and/or wherein the first, second, third, fourth, fifth and sixth optical waveguide (30, 34, 36, 38, 40, 42, 44, 48) together with the respective cannula end form the measuring tip (24) of the multispectral analysis probe (10). A multispectral analysis probe (10) as claimed in any preceding claim, wherein the optical fibers including the first, second, third, fourth, fifth, sixth and seventh fiber optic conductors (30, 34, 36, 38, 40, 42, 44, 48) and /or the light mixer conductor (45, 46), fiber optic conductors, in particular glass fiber lines or quartz fiber lines, and/or wherein the optical waveguides, including the first, second, third, fourth, fifth, sixth and seventh optical waveguide (30, 34, 36, 38 , 40, 42, 44, 48) and/or the light mixer conductor (45, 46), a diameter greater than or equal to 0.05 mm, in particular greater than or equal to 0.1 mm, and/or a diameter less than or equal to 1.0 mm, in particular less than or equal to 0.4 mm. A multispectral analysis probe (10) according to any one of the preceding claims, further comprising: a slidable analysis probe holder (12) for attaching the cannula (20) and for positioning the cannula (20) on the sample (100). Multispectral analysis probe (10) according to any one of the preceding claims, wherein the first optical waveguide (34) comprises a source end for connecting the first optical waveguide (34) to a first light source (80), and/or the second optical waveguide (36) comprises a source end for connecting the second optical waveguide (36) to a second light source (80), and/or the third optical fiber (38) includes a source end for connecting the third optical fiber (38) to a third light source (80). Multispectral analysis probe (10) according to any one of the preceding claims, wherein the fourth optical waveguide (40) comprises an output end for connecting the fourth optical waveguide (40) to a first evaluation device (70) and/or the fifth optical waveguide (42) comprises an output end for connecting the fifth optical waveguide (42) to the first or a second evaluation device (70) and/or the sixth optical waveguide (44) comprises an output end for connecting the sixth optical waveguide (44) to the first or a third evaluation device (70). Analysis system (9) with a multispectral analysis probe (10), in particular according to one of the preceding claims, for the analysis of different spectral ranges of a sample (100), comprising: a first optical source (80) for generating a first light pulse, a second optical source (80) for generating a second light pulse which is different from the first light pulse, the second light pulse being a light pulse exciting a Raman light pulse of the sample, a cannula (20) on a sample side of the multispectral analysis probe (10) for housing optical conductors (30, 34, 36, 38, 40, 42, 44, 45, 46, 48), the cannula comprising: at least one first optical waveguide (34) for transporting the first light pulse to the sample and a second optical waveguide (36) for transporting the light pulse exciting the one Raman light pulse of the sample to the sample, a measuring tip (24) on the sample side of the multispectral analysis probe (10), wherein the first optical source (80) and the second optical source (80) are connected to the cannula (20) to transmit the light pulses to the probe tip (24). Analysis system (9) with a multispectral analysis probe (10) according to one of the preceding claims, wherein the first light pulse of the first optical source (80) and the second light pulse of the second optical source (80) essentially simultaneously or in a short time interval one after the other over the cannula (20) to the sample (100) is emitted, so that the light pulses are essentially emitted onto the same sample material. Analysis system (9) with a multispectral analysis probe (10) according to any one of the preceding claims, further comprising: a first optical fiber (34) having a source end, the source end of the first optical fiber (34) being connected to the first light source (80); and/or a second optical fiber (36) having a source end, the source end of the second optical fiber (36) being connected to the second light source (80). Analysis system (9) with a multispectral analysis probe (10) according to one of the preceding claims, wherein the first optical source (80) is a fluorescent light source, a visual light source, in particular a halogen light source or a near-infrared light source. Analysis system (9) with a multispectral analysis probe (10) according to one of the preceding claims, wherein the first optical source (80) emits light in the wavelength range from 300 nm to 400 nm or in the wavelength range between 300 nm and 1200 nm, in particular between 700 and 1200 nm , or in the wavelength range between 1200 nm and 2200 nm. Analysis system (9) with a multispectral analysis probe (10) according to any one of the preceding claims, further comprising: a third optical source (80) for generating a third light pulse different from both the first light pulse and the second light pulse, and a third optical fiber (38) having a source end, the source end of the third optical fiber (38) being connected to the third light source (80). Analysis system (9) with a multispectral analysis probe (10) according to the preceding claim, wherein the third optical waveguide (38) couples the third light pulse into the light mixer (50) and the first, the second and the third light pulse via the common light mixer guide (45) through the cannula (20) for delivery to the sample (100). Analysis system (9) with a multispectral analysis probe (10) according to one of the preceding claims, wherein a fourth optical waveguide (40) is inserted into the cannula (20) in order to conduct the spectral response to the first light pulse from the cannula (20), and/or wherein a fifth optical waveguide (42) is inserted into the cannula (20) in order to conduct the spectral response to the second light pulse from the cannula (20), and/or wherein a sixth optical fiber (44) is inserted into the cannula (20) to direct the spectral response to the third light pulse from the cannula (20). Analysis system (9) with a multispectral analysis probe (10) according to any one of the preceding claims, further comprising: a first evaluation system (70) for spectral and/or photometric analysis of the spectral response to the first light pulse. Analysis system (9) with a multispectral analysis probe (10) according to the preceding claim, wherein the first evaluation system (70) is set up to also carry out a spectral and/or photometric analysis of the spectral response to the second and/or third light pulse. Analysis system (9) with a multispectral analysis probe (10) according to one of the preceding claims, wherein the optical waveguides (30, 34, 36, 38, 40, 42, 44, 45, 46, 48) comprise fiber optic conductors, in particular glass fiber lines or quartz fiber lines, and/or wherein the optical waveguides (30, 34, 36, 38, 40, 42, 44, 45, 46, 48) have a diameter of greater than or equal to 0.05 mm, in particular greater than or equal to 0.1 mm, and/or a diameter of less than or equal to 1, 0 mm, in particular less than or equal to 0.4 mm.

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